Nicotinic acetylcholine receptors (nAChRs) regulate critical physiological processes in the nervous and immune systems. These receptors represent potential therapeutic targets for new medicines to treat disorders of motor control (e.g., Parkinson's Disease), cognition (e.g., Alzheimer's Disease and schizophrenia), and sensory perception (e.g., chronic pain). Multiple subtypes of nAChRs exist, and different subtypes of these receptors underlie the pathophsyiology of various disease states. However, the ability to pharmacologically distinguish among these subtypes has been a problem. Conotoxins are unique peptides that not only are being used to overcome this problem but also serve as powerful probes for molecular characterization of nAChRs. We will utilize a newly discovered conotoxin RgIA, which specifically targets the alpha9alpha10 subtype of nAChR, to probe the molecular structure of the receptor. This subtype has a highly restricted tissue distribution and was recently shown to participate in a molecular pathway of neuropathic pain (i.e., pain induced by nerve injury). We will identify the features of RgIA that are responsible for its selectivity and high affinity for alpha9alpha10 nAChRs. We will use this information to create a battery of ligands, including fluorescent ones, to study alpha9alpha10 nAChRs in their native tissue environment. We have also exploited conotoxins to uncover and delineate previously unrecognized binding sites on the nAChR. In this regard, Project I will continue to work closely with discovery aspects of the Program to further characterize conotoxins that act at novel sites of nAChRs. Such investigation will enable further mechanistic understanding of the nAChR in particular and is expected to translate to improved understanding of ligandgated ion channels in general.